Outer membrane vesicles

ABSTRACT

The present invention provides an outer membrane vesicle (OMV) from a Gram-negative bacterium, comprising at least one heterologous protein that is free in the lumen of the OMV, wherein the OMV is capable of eliciting an immune response to the heterologous protein when administered to a mammal. The invention also provides methods for preparing the OMVs of the invention, pharmaceutical compositions comprising the OMVs of the invention, especially immunogenic compositions and vaccines, and methods of generating an antibody immune response in a mammal using OMVs.

This application claims the benefit of U.S. provisional application61/702,296 filed Sep. 18, 2012, and of the U.S. provisional application61/799,311 filed Mar. 15, 2013, the complete contents of all of whichare hereby incorporated herein by reference for all purposes.

TECHNICAL FIELD

This invention relates to vesicles from Gram-negative bacteria. Thevesicles comprise heterologous proteins in their lumens. The vesiclesare particularly useful in immunogenic compositions, e.g. vaccines.

BACKGROUND ART

Gram-negative bacteria can spontaneously release outer membrane vesicles(OMVs) during growth due to the turgor pressure of the cell envelope.The formation of such OMVs can be facilitated by disruption of certainbacterial components e.g. references 1 and 2 disrupted the E. coliTol-Pal system to provide strains which release vesicles into theculture medium during growth. OMVs can also be produced by disruption ofwhole bacteria. Known OMV production methods include methods which usedetergent treatment (e.g. with deoxycholate) [3 & 4], detergent-freemethods [5], or sonication [6], etc.

OMVs are rich in immunogenic cell surface-associated, periplasmic andsecreted antigens and have been used as vaccines, e.g. against Neisseriameningitidis serogroup B [7]. They are particularly suited for this usebecause the vesicles contain compounds that act as adjuvants, elicitingstrong immune responses against the antigens. In this way, the vesiclesare a closer mimic of the native bacterium for the immune system thanpurified antigenic proteins or other bacterial components. OMVstherefore remain an attractive target for vaccines and other immunogeniccompositions. It has been suggested that the immunogenic properties ofsome protein antigens can be increased by engineering OMVs to displaymultiple antigens on the surfaces of OMVs by using ClyA as a fusionpartner [8].

Several attempts have been made to target heterologous proteins, and inparticular heterologous antigens, to OMVs. However, to date antigensthat are foreign to the parental bacteria remain notably absent fromOMVs largely because of challenges associated with the transport ofheterologous proteins to the vesicles [11]. Most attempts to targetheterologous proteins to OMVs have relied on covalent linkage of theheterologous proteins to integral membrane proteins. Examples of suchcovalently-linked heterologous proteins include fusions of the FLAGepitope to the full-length sequence of OmpA (outer membrane protein A),fusions of the FLAG epitope to the full-length sequence of PagP(PhoPQ-activated gene P) [9], and fusions of GFP to ClyA (Cytolysin)[10]. By virtue of their covalent linkages to membrane proteins, theresulting fusion proteins are targeted to the outer membrane and arethus included in the OMVs. These methods have drawbacks, in particularbecause it is difficult to overexpress a large amount of an integralmembrane protein without detrimental effects of the transformedbacterium.

Targeting periplasmic proteins to OMVs has also proven to be difficult.A fusion of GFP to a Tat (twin arginine transporter) signal sequenceresulted in overexpression of GFP that was targeted to the periplasm,but GFP fluorescence was barely above background fluorescence levels inOMVs [11], suggesting that the GFP was either not incorporated into theOMVs or was non-functional in the OMV because of incorrect folding.

There remains a need to develop a method suitable for expressingheterologous proteins in OMVs, and in particular a method to expressantigenic proteins in OMVs. There also remains a need for alternative orimproved OMVs, particularly for use in vaccines.

DISCLOSURE OF THE INVENTION

The inventors have discovered that targeting heterologous proteins tothe lumen of OMVs overcomes many of the problems associated withtargeting heterologous proteins to the membrane of OMVs. Surprisingly,the inventors have also found that OMVs containing heterologous proteinsthat are in the lumen are capable of eliciting immune responses to theproteins when administered to a mammal.

Thus, the invention provides an outer membrane vesicle (OMV) from aGram-negative bacterium, wherein the OMV comprises at least oneheterologous protein that is free in its lumen, and the OMV is capableof eliciting an immune response to the heterologous protein whenadministered to a mammal.

The invention also provides a method of preparing an OMV of theinvention, the method comprising the step of expressing the heterologousprotein in the periplasm of the Gram-negative bacterium. The inventionfurther provides an OMV obtained or obtainable by this method.

The invention also provides a pharmaceutical composition comprising (a)an OMV of the invention and (b) a pharmaceutically acceptable carrier.

The invention also provides a method of generating an immune response ina mammal, the method comprising administering an effective amount of anOMV from a Gram-negative bacterium to the mammal, wherein the OMVcomprises at least one heterologous protein in its lumen, and whereinthe immune response is to the heterologous protein in the OMV. In someembodiments of this aspect of the invention, the protein is free in thelumen of the OMV. The invention also provides a method of generating animmune response in a mammal comprising administering a pharmaceuticalcomposition of the invention to the mammal, wherein the immune responseis to the heterologous protein in the OMV.

OMVs

The present invention provides an outer membrane vesicle (OMV) from aGram-negative bacterium, wherein the OMV comprises at least oneheterologous protein that is free in its lumen, and the OMV is capableof eliciting an immune response to the heterologous protein whenadministered to a mammal.

OMVs are well known in the art and are spontaneously released intoculture medium by bacteria. ‘Native OMVs’ (‘NOMVs’ [12]), microvesicles(MVs [13]), detergent-extracted OMVs (DOMVs), mutant-derived OMVs(m-OMV), and blebs, which are outer-membrane protrusions that remainattached to bacteria prior to release as MVs ([14]; [15]), all form partof the invention and are collectively referred to as OMVs herein.

OMVs of the invention can be obtained from any suitable Gram-negativebacterium. The Gram-negative bacterium is typically E. coli. However,instead of E. coli it may be a different Gram-negative bacterium.Preferred Gram-negative bacteria for use in the invention includebacteria that are not pathogenic in humans. For example, the bacteriamay be commensalistic in humans. However, in some embodiments bacteriaare used that are not typically found in human hosts at all. Exemplaryspecies for use in the invention include species in any of generaEscherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia,Brucella, Chlamydia Haemophilus, Legionella, Pseudomonas, Yersinia,Helicobacter, Salmonella, Vibrio, etc. In particular, the bacterium maybe a Shigella species (such as S. dysenteriae, S. flexneri, S. boydii orS. sonnei). Alternatively, it may be a Neisseria species, particularly anon-pathogenic species such as N. bacilliformis, N. cinerea, N.elongata, N. flavescens, N. lactamica, N. macacae, N. mucosa, N.polysaccharea, N. sicca or N. subflava, and in particular N. lactamica.Alternatively, a pathogenic species of Neisseria may be used, e.g. N.gonorrhoeae or N. meningitidis. In other examples, the bacterium may beBordetella pertussis, Borrelia burgdorferi, Brucella melitensis,Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxellacatarrhalis, Haemophilus influenzae (including non-typeable stains),Legionella pneumophila, Pseudomonas aeruginosa, Yersinia enterocolitica,Helicobacter pylori, Salmonella enterica (including serovars typhi andtyphimurium, as well as serovars paratyphi and enteritidis), Vibriocholerae, Proteus, Citrobacter, Serratia, Erwinia, Pasteurella etc.Photosynthetic Gram-negative bacteria may also be used. Typically, thebacterium is a competent strain. This feature facilitates geneticmodification of the bacterium.

In a particular embodiment, the Gram-negative bacterium is a“hyperblebbing” strain of that bacterium. Hyperblebbing Gram-negativebacteria from which blebs may more easily be made in higher yield andmay be more homogeneous in nature are described in WO 02/062378. Forexample, the blebs may be derived from bacteria selected from the groupconsisting of Neisseria meningitidis, Neisseria lactamica, Neisseriagonorrhoeae, Helicobacter pylori, Salmonella typhi, Salmonellatyphimurium, Vibrio cholerae, Shigella spp., Haemophilus influenzae,Bordetella pertussis, Pseudomonas aeruginosa and Moraxella catarrhalis.

In a specific embodiment, the bacterium is an E. coli ompA mutant and/orE. coli tolR mutant. In some embodiments, the bacterium is selected fromE. coli BL21(DE3)ΔompA, E. coli BL21(DE3)ΔompAΔtolR, E. coliBL21(DE3)ΔtolR, E. coli ΔnlpI, or E. coli ΔdegP. The A symbol is usedherein to refer to a bacterial strain from which the coding sequence ofthe gene recited after the A symbol has been deleted. Thus, a bacterialstrain which is “ΔompA” does not comprise the coding sequence for theompA gene. Likewise, a bacterial strain which is “ΔtolR” does notcomprise the coding sequence for the tolR gene. The entire codingsequence may be deleted. However, the coding sequence may alternativelybe deleted in part. For example, the N-terminal half or the C-terminalhalf may be deleted. Alternatively, the ompA and/or tolR genes may bemutated by the introduction of one or more substitutions and/orinsertions.

The E. coli ΔtolR mutant strains and E. coli ΔompA mutant strainsoverproduce OMVs relative to wild type E. coli. Thus, the mutation ofthe ompA gene and/or one or more components of the Tol-Pal complexresults in the mutant bacterium producing an increased number of OMVscompared to its respective wild type strain which carries a wild typeompA gene and/or Tol-Pal complex. OmpA is an integral membrane proteinand is the most abundant of the outer membrane proteins in E. coli. Itis, therefore, surprising that an E. coli lacking the OmpA protein isviable. Indeed, according to Murakami et al. [16], an E. coli ompAsingle mutant cannot promote vesicle release.

Heterologous Protein

The heterologous protein of the invention is targeted to and expressedin the periplasm of the Gram-negative bacterium such that theheterologous protein is in the lumen of the OMV. In some embodiments,the heterologous protein is free in the lumen of the OMV.

The protein may be an amino acid polymer of any length. The amino acidpolymer may be linear or branched, it may comprise modified amino acids,and it may be interrupted by non-amino acids. The terms also encompassan amino acid polymer may have been modified naturally or byintervention; for example, by disulfide bond formation, additionalglycosylation, partial or complete deglycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labelling component. Also included within thedefinition are, for example, proteins containing one or more analoguesof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Proteins can occur assingle chains or associated chains. Proteins in the context of theinvention can be naturally or non-naturally glycosylated (i.e. thepolypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringpolypeptide).

As used herein, the term “heterologous” means that the protein is from aspecies that is different from the species of bacterium from which theOMV is obtained (the heterologous organism). Typically, the protein isan antigen from a pathogen genus different from the genus of bacteriumfrom which the OMV is obtained.

In a specific embodiment of the invention, the heterologous protein isan immunogenic protein which can elicit an immune response in therecipient. In a specific embodiment, the immunogenic protein, and thusthe heterologous protein, comprises or consists of an antigen. Theantigen may elicit an immune response against a protist, a bacterium, avirus, a fungus, or any other pathogen including multicellularpathogens, or a parasite (or, in some embodiments, against an allergen;and in other embodiments, against a tumor antigen). The immune responsemay comprise an antibody response (usually including IgG) and/or acell-mediated immune response. The polypeptide antigen will typicallyelicit an immune response which recognises the corresponding bacterial,viral, fungal or parasite (or allergen or tumour) polypeptide, but insome embodiments the polypeptide may act as a mimotope to elicit animmune response which recognises a bacterial, viral, fungal or parasitesaccharide. The antigen will typically be a surface polypeptide e.g. anadhesin, a hemagglutinin, an envelope glycoprotein, a spikeglycoprotein, etc.

In some embodiments the antigen elicits an immune response against oneof these bacteria:

-   Neisseria meningitidis: useful antigens include, but are not limited    to, membrane proteins such as adhesins, autotransporters, toxins,    iron acquisition proteins, factor H binding protein (fHbp or 741),    Neisseria Heparin-Binding Antigen (NHBA or 287), NadA (or 961),    953/936 and Neisseria meningitides serogroup B by (fHbp). A    combination of three useful polypeptides is disclosed in reference    17.-   Streptococcus pneumoniae: useful polypeptide antigens are disclosed    in reference 18. These include, but are not limited to, the RrgB    pilus subunit, the beta-N-acetyl-hexosaminidase precursor (spr0057),    spr0096, General stress protein GSP-781 (spr2021, SP2216),    serine/threonine kinase StkP (SP1732), and pneumococcal surface    adhesin PsaA.-   Streptococcus pyogenes: useful antigens include, but are not limited    to, the polypeptides disclosed in references 19 and 20, e.g.    GAS25-574, such as GAS 25 (SEQ ID:41, SEQ ID:42), GAS40 (SEQ ID:43,    SEQ ID:44, SEQ ID:45, SEQ ID:46, SEQ ID:47, SEQ ID:48, SEQ ID:49,    SEQ ID:50, SEQ ID:51, SEQ ID:52, SEQ ID:53, SEQ ID:54, SEQ ID:55,    SEQ ID:56, SEQ ID: 57, SEQ ID:58, SEQ ID:59, SEQ ID:60, SEQ ID:61,    SEQ ID:62, SEQ ID:63, SEQ ID:64, SEQ ID:65, SEQ ID:66, SEQ ID: 67,    SEQ ID:68, SEQ ID:69, SEQ ID:70), GAS57 (SEQ ID:39, SEQ ID:40, SEQ    ID:71, SEQ ID:72, SEQ ID:73, SEQ ID:74; SEQ ID:75),88, 23, 99, 97,    24, 5, 208, 193, 67, 64, 101, 205, 268, 68, 189, 165 or 201.-   Moraxella catarrhalis.-   Bordetella pertussis: Useful pertussis antigens include, but are not    limited to, acellular or whole-cell pertussis antigens, pertussis    holotoxin or toxoid (PT), filamentous haemagglutinin (FHA),    pertactin, and agglutinogens 2 and 3.-   Staphylococcus aureus: Useful antigens include, but are not limited    to, the polypeptides disclosed in reference 21, such as a hemolysin,    esxA, esxB, esxAB, ferrichrome-binding protein (sta006) and/or the    sta011 lipoprotein.-   Clostridium tetani: the typical antigen is tetanus toxoid.-   Cornynebacterium diphtheriae: the typical antigen is diphtheria    toxoid.-   Haemophilus influenzae: Useful antigens include, but are not limited    to, the polypeptides disclosed in references 22 and 23.-   Pseudomonas aeruginosa-   Streptococcus agalactiae: useful antigens include, but are not    limited to, the polypeptides disclosed in reference 19, such as 67,    80, 1523, 3, 328 or 211.-   Chlamydia trachomatis: Useful antigens include, but are not limited    to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS,    CT547, Eno, HtrA and MurG (e.g. as disclosed in reference 24. LcrE    [25] and HtrA [26] are two preferred antigens.-   Chlamydia pneumoniae: Useful antigens include, but are not limited    to, the polypeptides disclosed in reference 27.-   Helicobacter pylori: Useful antigens include, but are not limited    to, CagA, VacA, NAP, and/or urease[28].-   Escherichia coli: Useful antigens include, but are not limited to,    antigens derived from enterotoxigenic E. coli (ETEC),    enteroaggregative E. coli (EAggEC), diffusely adhering E. coli    (DAEC), enteropathogenic E. coli (EPEC), extraintestinal    pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC).    ExPEC strains include uropathogenic E. coli (UPEC) and    meningitis/sepsis-associated E. coli (MNEC). Useful UPEC polypeptide    antigens are disclosed in references 29 and 30. Useful MNEC antigens    are disclosed in reference 31. A useful antigen for several E. coli    types is AcfD [32].-   Bacillus anthracis-   Yersinia pestis: Useful antigens include, but are not limited to,    those disclosed in references 33 and 34.-   Staphylococcus epidermidis, e.g. type I, II and/or III capsular    polysaccharide obtainable from strains ATCC-31432, SE-360 and SE-10-   Clostridium perfringens or Clostridium botulinums-   Legionella pneumophila-   Coxiella burnetii-   Brucella, such as B. abortus, B. canis, B. melitensis, B.    neotomae, B. ovis, B. suis, B. pinnipediae.-   Francisella, such as F. novicida, F. philomiragia, F. tularensis.-   Neisseria gonorrhoeae-   Treponema pallidum-   Haemophilus ducreyi-   Enterococcus faecalis or Enterococcus faecium-   Staphylococcus saprophyticus-   Yersinia enterocolitica-   Mycobacterium tuberculosis-   Mycobacterium leprae-   Rickettsia-   Listeria monocytogenes-   Vibrio cholerae-   Salmonella typhi-   Borrelia burgdorferi-   Porphyromonas gingivalis-   Klebsiella-   Rickettsia prowazekii.

In some embodiments the antigen is an antigen from Chlamydia,Streptococcus, Pseudomonas, Shigella, Campylobacter, Salmonella,Neisseria or Helicobacter.

In some embodiments the antigen elicits an immune response against oneof these viruses:

-   Orthomyxovirus: Useful antigens can be from an influenza A, B or C    virus, such as the hemagglutinin, neuraminidase or matrix M2    proteins. Where the antigen is an influenza A virus hemagglutinin it    may be from any subtype e.g. H1, H2, H3, H4, H5, H6, H7, H8, H9,    H10, H11, H12, H13, H14, H15 or H16.-   Paramyxoviridae viruses: Viral antigens include, but are not limited    to, those derived from Pneumoviruses (e.g. respiratory syncytial    virus, RSV), Rubulaviruses (e.g. mumps virus), Paramyxoviruses (e.g.    parainfluenza virus), Metapneumoviruses and Morbilliviruses (e.g.    measles virus).-   Poxviridae: Viral antigens include, but are not limited to, those    derived from Orthopoxvirus such as Variola vera, including but not    limited to, Variola major and Variola minor.-   Picornavirus: Viral antigens include, but are not limited to, those    derived from Picornaviruses, such as Enteroviruses, Rhinoviruses,    Heparnavirus, Cardioviruses and Aphthoviruses. In one embodiment,    the enterovirus is a poliovirus e.g. a type 1, type 2 and/or type 3    poliovirus. In another embodiment, the enterovirus is an EV71    enterovirus. In another embodiment, the enterovirus is a coxsackie A    or B virus.-   Bunyavirus: Viral antigens include, but are not limited to, those    derived from an Orthobunyavirus, such as California encephalitis    virus, a Phlebovirus, such as Rift Valley Fever virus, or a    Nairovirus, such as Crimean-Congo hemorrhagic fever virus.-   Heparnavirus: Viral antigens include, but are not limited to, those    derived from a Heparnavirus, such as hepatitis A virus (HAV) e.g.    inactivated virus, hepatitis B virus e.g. the surface and/or core    antigens or hepatitis C virus.-   Filovirus: Viral antigens include, but are not limited to, those    derived from a filovirus, such as an Ebola virus (including a Zaire,    Ivory Coast, Reston or Sudan ebolavirus) or a Marburg virus.-   Togavirus: Viral antigens include, but are not limited to, those    derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an    Arterivirus. This includes rubella virus.-   Flavivirus: Viral antigens include, but are not limited to, those    derived from a Flavivirus, such as Tick-borne encephalitis (TBE)    virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever virus,    Japanese encephalitis virus, Kyasanur Forest Virus, West Nile    encephalitis virus, St. Louis encephalitis virus, Russian    spring-summer encephalitis virus, Powassan encephalitis virus.-   Pestivirus: Viral antigens include, but are not limited to, those    derived from a Pestivirus, such as Bovine viral diarrhea (BVDV),    Classical swine fever (CSFV) or Border disease (BDV).-   Hepadnavirus: Viral antigens include, but are not limited to, those    derived from a Hepadnavirus, such as Hepatitis B virus. A    composition can include hepatitis B virus surface antigen (HBsAg).-   Other hepatitis viruses: A composition can include an antigen from a    hepatitis C virus, delta hepatitis virus, hepatitis E virus, or    hepatitis G virus.-   Rhabdovirus: Viral antigens include, but are not limited to, those    derived from a Rhabdovirus, such as a Lyssavirus (e.g. a Rabies    virus) and Vesiculovirus (VSV). An example of a Rabies antigen is    lyophilised inactivated virus.-   Caliciviridae: Viral antigens include, but are not limited to, those    derived from Calciviridae, such as Norwalk virus (Norovirus), and    Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.-   Coronavirus: Viral antigens include, but are not limited to, those    derived from a SARS coronavirus, avian infectious bronchitis (IBV),    Mouse hepatitis virus (MHV), and Porcine transmissible    gastroenteritis virus (TGEV). The coronavirus antigen may be a spike    polypeptide.-   Retrovirus: Viral antigens include, but are not limited to, those    derived from an Oncovirus, a Lentivirus (e.g. HIV-1 or HIV-2) or a    Spumavirus, e.g. gp120, gp140 or gp160-   Reovirus: Viral antigens include, but are not limited to, those    derived from an Orthoreovirus, a Rotavirus, an Orbivirus, or a    Coltivirus.

Parvovirus: Viral antigens include, but are not limited to, thosederived from Parvovirus B19.

-   Herpesvirus: Viral antigens include, but are not limited to, those    derived from a human herpesvirus, such as, by way of example only,    Herpes Simplex Viruses (HSV) (e.g. HSV types 1 and 2),    Varicella-zoster virus (VZV), Epstein-Barr virus (EBV),    Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus    7 (HHV7), and Human Herpesvirus 8 (HHV8).-   Papovaviruses: Viral antigens include, but are not limited to, those    derived from Papillomaviruses and Polyomaviruses. The (human)    papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18,    31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one or    more of serotypes 6, 11, 16 and/or 18.-   Adenovirus: Viral antigens include those derived from adenovirus    serotype 36 (Ad-36).

In some embodiments, the antigen elicits an immune response against avirus which infects fish, such as: infectious salmon anemia virus(ISAV), salmon pancreatic disease virus (SPDV), infectious pancreaticnecrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystisdisease virus (FLDV), infectious hematopoietic necrosis virus (IHNV),koi herpesvirus, salmon picorna-like virus (also known as picorna-likevirus of atlantic salmon), landlocked salmon virus (LSV), atlanticsalmon rotavirus (ASR), trout strawberry disease virus (TSD), cohosalmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).

Fungal antigens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowii,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia,Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi;the less common are Brachiola spp, Microsporidium spp., Nosema spp.,Pleistophora spp., Trachipleistophora spp., Vittaforma sppParacoccidioides brasiliensis, Pneumocystis carinii, Pythiumninsidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomycesboulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrixschenckii, Trichosporon beigelii, Toxoplasma gondii, Penicilliummarneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrixspp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp,Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

In some embodiments the antigen elicits an immune response against aparasite from the Plasmodium genus, such as P. falciparum, P. vivax, P.malariae or P. ovale. Thus the invention may be used for immunisingagainst malaria. In some embodiments the antigen elicits an immuneresponse against a parasite from the Caligidae family, particularlythose from the Lepeophtheirus and Caligus genera e.g. sea lice such asLepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments the antigen elicits an immune response against:pollen allergens (tree-, herb, weed-, and grass pollen allergens);insect or arachnid allergens (inhalant, saliva and venom allergens, e.g.mite allergens, cockroach and midges allergens, hymenopthera venomallergens); animal hair and dandruff allergens (from e.g. dog, cat,horse, rat, mouse, etc.); and food allergens (e.g. a gliadin). Importantpollen allergens from trees, grasses and herbs are such originating fromthe taxonomic orders of Fagales, Oleales, Pinales and platanaceaeincluding, but not limited to, birch (Betula), alder (Alnus), hazel(Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria andJuniperus), plane tree (Platanus), the order of Poales including grassesof the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris,Secale, and Sorghum, the orders of Asterales and Urticales includingherbs of the genera Ambrosia, Artemisia, and Parietaria. Other importantinhalation allergens are those from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and thosefrom mammals such as cat, dog and horse, venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (Apidae), wasps(Vespidea), and ants (Formicoidae).

In some embodiments the antigen is a tumor antigen selected from: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE,BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which canbe used, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors; (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-abl (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT; (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), mammaglobin, alpha-fetoprotein (associated with, e.g.,hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin(associated with, e.g., pancreatic and gastric cancer), telomerasecatalytic protein, MUC-1 (associated with, e.g., breast and ovariancancer), G-250 (associated with, e.g., renal cell carcinoma), p53(associated with, e.g., breast, colon cancer), and carcinoembryonicantigen (associated with, e.g., breast cancer, lung cancer, and cancersof the gastrointestinal tract such as colorectal cancer); (d) sharedantigens, for example, melanoma-melanocyte differentiation antigens suchas MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma); (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer; (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example). In certainembodiments, tumor antigens include, but are not limited to, p15,Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virusantigens, EBNA, human papillomavirus (HPV) antigens, including E6 andE7, hepatitis B and C virus antigens, human T-cell lymphotropic virusantigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4,791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM),HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16,TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6,TAG72, TLP, TPS, and the like.

In a further specific example, the heterologous protein is β lactamase(TEM1), fHbp from Neisseria meningitides, the double mutant ofextracellular cholesterol depending streptolysin O (Slo-dm) fromStreptococcus pyogenes, the cell envelope serine protease SpyCep fromStreptococcus pyogenes, or the putative surface exclusion proteinSpy0269 from Streptococcus pyogenes.

The heterologous protein may be a soluble protein, a peripheral membraneprotein or an integral membrane protein when expressed in theheterologous organism from which it is derived, i.e. when present in itsnative environment. For example, if the heterologous protein is derivedfrom a Gram-negative bacterium, it may be a cytoplasmic, periplasmic ormembrane-associated protein in the native Gram-negative bacterium.However, when present in the OMV, the heterologous protein is in thelumen of the OMV, and preferably free in the lumen of the OMV.Therefore, the heterologous protein may be modified, as compared to thewild-type protein, for example by deleting any membrane anchor(s).

The term “in the lumen” of the OMV encompasses both proteins that aremembrane associated but not surface exposed, and proteins that are freein the lumen of the OMV. The heterologous protein is generally free inthe lumen of the OMV in the present invention. By “free in the lumen” itis meant that the heterologous protein is not integrally associated withthe membrane of the OMV. Integral association with the membranedescribes those proteins that require the use of a detergent or otherapolar solvent to dissociate the protein from the membrane. A review ofmembrane anchors for integral association with the membrane can be foundin reference 35. A protein that is free in the lumen of the OMV may beassociated with the membrane or an integral membrane protein bynon-covalent interactions or may not associate with the membrane of theOMV at all. For example, the protein may loosely or temporarilyassociate with the membrane, e.g. via hydrophobic, electrostatic, ionicand/or other non-covalent interactions with the lipid bilayer and/or toan integral protein.

One advantage of the heterologous protein being in the lumen of OMV,rather than being associated with the membrane and exposed, is that itmay be protected from protease degradation in vivo. This protection mayin turn result in more efficient B cell activation.

In a particular embodiment, the heterologous protein is a solubleprotein. By “soluble protein” it is meant that the protein does not formany association with lipid membrane. A soluble protein does not containa membrane anchor such as a peptide transmembrane domain, other peptidemembrane anchoring domain, or a non-peptide membrane anchor such as alipid.

The OMV is capable of eliciting an immune response to the heterologousprotein when administered to a mammal. The immune response may be acellular or a humoral immune response. Typically, the immune response isan antibody response.

In one embodiment, the OMV of the invention is capable of eliciting animmune response against the pathogen from which the heterologous proteinis derived. For example, the heterologous protein preferably elicits aT-cell immune response that can neutralise the infection and/orvirulence of the pathogen from which the heterologous protein isderived. Preferred heterologous proteins for use in the invention aretherefore those which are recognised by the cellular immune system uponinfection with a pathogen of interest. More preferred are thoseheterologous proteins which elicit a protective T-cell immune responseagainst a pathogen of interest.

In one embodiment, the OMV of the invention is capable of elicitingantibodies that recognise a pathogen from which the heterologous proteinis derived. For example, the heterologous protein preferably elicitsantibodies that can bind to, and preferably neutralise the infectionand/or virulence of the pathogen from which the heterologous protein isderived. Preferred heterologous proteins for use in the invention aretherefore those which are recognised by anti-sera upon infection with apathogen of interest. More preferred are those heterologous proteinswhich elicit a protective immune response against a pathogen ofinterest.

In some embodiments, the heterologous protein is immunogenic when it ispresented in the OMV but is not immunogenic when administered inpurified form.

In one embodiment, the heterologous proteins of the invention arefunctionally active in the lumen of the OMV and/or upon release from thelumen of the OMV (e.g. by detergent-mediated disruption of the OMV).Functional activity is an indicator that the heterologous protein isfolded correctly and has the same or substantially the same tertiary andquaternary structure as the same protein in its native state. By“functionally active” it is meant that the heterologous protein retainsat least 50% or more of at least one biological activity of the sameprotein when expressed in its native environment (e.g. in the organismfrom which it is derived). For example, the heterologous protein can beconsidered to be functionally active if it retains at least 50%, 60%,70%, 80%, 90% or more or of at least one biological activity of the sameprotein when expressed in its native environment.

In embodiments in which the heterologous protein comprises or consistsof a fragment of a wild type protein or of a variant thereof, thefragment or variant may be functionally active. By “fragment of a wildtype protein” it is meant that the heterologous protein comprises orconsists of at least 7 consecutive amino acids from the wild typeprotein. In some embodiments, the fragment consists of at least 7, 8, 9,10, 20, 30, 40 or more amino acids from the wild type protein. Thefragment may consist of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more of the wild type protein.

Preferably, the fragment is an immunogenic fragment of the heterologousprotein. By “immunogenic fragment” it is meant that the fragment has atleast one epitope in common with the heterologous protein. The term“epitope” encompasses any kind of epitope and includes both B-cell andT-cell epitopes, and both linear and discontinuous epitopes. In oneembodiment, an antibody that binds specifically to the heterologousprotein also binds specifically to the immunogenic fragment, i.e. theheterologous protein and immunogenic fragment thereof both contain theepitope to which that antibody binds. By “binds specifically”, it ismeant that the antibodies bind to the heterologous protein of theinvention with substantially greater affinity than to BSA. Preferably,the affinity is at least 100-fold, 10³-fold, 10⁴-fold, 10⁵-fold,10⁶-fold etc. greater for the heterologous protein of the invention thanfor BSA.

Epitopes present in heterologous proteins can be determined and/orpredicted using any methods known in the art. For example, epitiopeprediction software such as EpiToolKit, which is a web server forcomputational immunomics [36]. This epitope prediction software providesseveral methods for predicting potential T-cell eptiopes, both for MHCClass I and MHC Class II binding epitopes.

The presence of B-cell epitopes can also be predicted using any knownmethod in the art, for example as described in references [37, 38 and39]. The presence of continuous linear epitopes and/or discontinuousepitopes can be predicted using the methods described therein.

By “variant of a wild type protein” it is meant that the heterologousprotein comprises or consists of a full length protein, e.g. a proteinwith the same number of amino acids as the wild-type protein, or afragment of the wild type protein that contains one or more variationsin amino acid sequence when compared to the wild type sequence. Avariant may have at least 50%, 60%, 70%, 80%, 90%, 95% or more sequenceidentity to the wild type protein. In some embodiments, the variant isalso functionally active.

The heterologous protein may be fused to a fusion partner, i.e. theheterologous protein may be part of a fusion protein. Fusion proteinsmay comprise a sequence -X-Y- or -Y-X-, wherein: -X- is a heterologousprotein as defined above, and -Y- is an additional polypeptide sequence.In one particular Embodiment -Y- is a protein tag that aids detection ofthe heterologous protein such as 6×HIS, FLAG, HA, GST, GFP or anotherfluorescent protein, and/or luciferase or any suitable polypeptide whichaids in the function of the heterologous protein. When the heterologousprotein is part of a fusion protein, the entire fusion protein will bein the lumen of the OMV. In some embodiments, the fusion protein will befree in the lumen of the OMV.

The OMV of the present invention comprises at least one heterologousprotein in the lumen of the OMV. The OMV may therefore contain at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous proteins in the lumenof the OMV, and preferably free in the lumen of the OMV. In addition tothe at least one heterologous protein in the lumen of the OMV, the OMVof the invention may also comprise at least one heterologous proteinassociated with the membrane of the OMV. For example, the OMV of theinvention may comprise at least one heterologous protein in the lumen ofthe OMV and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous proteinsassociated with the membrane of the OMV.

In a particular embodiment, the heterologous proteins comprise thedouble mutant of extracellular cholesterol depending streptolysin 0(Slo-dm) from Streptococcus pyogenes and the putative surface exclusionprotein Spy0269 from Streptococcus pyogenes. In a further particularembodiment, the heterologous proteins comprise the double mutant ofextracellular cholesterol depending streptolysin 0 (Slo-dm) fromStreptococcus pyogenes, the cell envelope serine protease SpyCEP fromStreptococcus pyogenes, and the putative surface exclusion proteinSpy0269 from Streptococcus pyogenes.

Methods of Preparing OMVs of the Invention

The invention also provides a method of preparing an OMV of theinvention, the method comprising the step of expressing the heterologousprotein in the periplasm of the Gram-negative bacterium.

In a particular embodiment, the heterologous protein is expressed in theperiplasm of the Gram-negative bacterium using an expression vectorcomprising a nucleic acid sequence encoding the heterologous proteinoperatively linked to a nucleic acid encoding a signal sequence of aperiplasmic protein.

Targeting of the heterologous proteins can be achieved by fusing thesignal sequence of a protein which is naturally found in the periplasmand/or OMVs to a heterologous protein. Protein translocation through theinner membrane and to the periplasm may, for example, be accomplished byway of one of three pathways: SecB-dependent (SEC), signal recognitionparticle (SRP) or twin-arginine translocation (TAT). Any of thesepathways can be used.

An example of a periplasmic signal sequence that can be used in thepresent invention is the signal sequence of OmpA. However, otherpossible signal sequences could be used including the Tat signalsequence, and the DsbA signal sequence. Export to the periplasm can beoptimised using a series of vectors, each targeting a different exportpathway. For example, the ACES Signal Sequence Expression Vectors [40]can be used to optimise translocation of the heterologous protein to theperiplasm.

In some embodiments, the native signal sequence of the heterologousprotein is replaced by the signal sequence of a periplasmic protein. Inother embodiments, the heterologous protein is fused to the signalsequence of the periplasmic protein without replacing the native signalsequence, if present.

Specific embodiments of this aspect of the invention include using anexpression vector comprising a nucleic acid sequence encoding the signalsequence of OmpA operably linked to a nucleic acid encoding theheterologous protein, for example the pET-OmpA plasmid shown in FIG. 1.In this embodiment, the OmpA signal sequence may have the nucleotidesequence ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC (SEQ ID NO:1). In this embodiment, the plasmid is a pET21b-derivedplasmid. However, any other suitable plasmid backbone known in the artcan also be used. Suitable plasmid backbones include pGEX, pUC19, pALTR,pET, pQE, pLEX, pHAT or any other plasmid vector that is cablable ofreplication in gram-negative bacteria.

Any Gram-negative bacteria that are capable of producing OMVs, forexample those mentioned herein, can be transformed with the expressionvectors described above in order to produce OMVs comprising theheterologous protein in their lumen, and preferably free in their lumen

Methods for preparing OMVs are known in the art, and any suitable methodcan be used to generate OMVs of the invention. These methods generallyinvolve a step of obtaining vesicles from a culture of the bacterium.The OMVs can be obtained by disruption of or blebbing from the outermembrane of the bacterium to form vesicles therefrom. OMVs can also beprepared artificially from bacteria, for example by sarkosyl-extractionof OMVs from ‘ΔGNA33’ meningococci, as described in reference 41.‘Native OMVs’ (‘NOMVs’ [42]), microvesicles (MVs [43]),detergent-extracted OMVs (DOMVs), mutant-derived OMVs (m-OMV), andblebs, which are outer-membrane protrusions that remain attached tobacteria prior to release as MVs ([44]; [45]), all form part of theinvention and are collectively referred to as OMVs herein.

OMVs (including blebs, MVs and NOMVs) include naturally-occurringmembrane vesicles that form spontaneously during bacterial growth andare released into culture medium. Preferably, the OMVs of the inventionare naturally occurring OMVs because separation ofspontaneously-released OMVs from the culture medium is more convenientthan methods which involve deliberate disruption of the outer membrane(e.g. by detergent treatment or sonication) to produce artificiallyinduced OMVs. Moreover, they are substantially free from inner membraneand cytoplasmic contamination. OMVs typically have a diameter of 35-120nm by electron microscopy e.g. 50 nm diameter and can be purified fromthe culture medium. The purification ideally involves separating theOMVs from living and/or intact bacteria e.g. by size-based filtrationusing a filter, such as a 0.22 μm filter, which allows the OMVs to passthrough but which does not allow intact bacteria to pass through, or byusing low speed centrifugation to pellet cells while leaving the blebsin suspension. A preferred method involving a two stage size filtrationprocess is described in ref 46.

Thus, unlike the culture medium, OMV-containing compositions of theinvention will generally be substantially free from whole bacteria,whether living or dead. The size of the OMVs means that they can readilybe separated from whole bacteria by filtration e.g. as typically usedfor filter sterilisation. Although OMVs will pass through a standard0.22 μm filters, these can rapidly become clogged by other material, andso it may be useful to perform sequential steps of filter sterilisationthrough a series of filters of decreasing pore size before using a 0.22μm filter. Examples of preceding filters would be those with pore sizeof 0.8 μm, 0.45 μm, etc.

In an alternative embodiment, OMVs may be prepared artificially frombacteria, and may be prepared using detergent treatment (e.g. withdeoxycholate or sarkosyl), or by non-detergent means (e.g. see reference47). Techniques for forming OMVs include treating bacteria with a bileacid salt detergent (e.g. salts of lithocholic acid, chenodeoxycholicacid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholicacid, etc., with sodium deoxycholate[48 & 49] being preferred fortreating Neisseria) at a pH sufficiently high not to precipitate thedetergent [50]. Other techniques may be performed substantially in theabsence of detergent [47] using techniques such as sonication,homogenisation, microfluidisation, cavitation, osmotic shock, grinding,French press, blending, etc. Methods using no or low detergent canretain useful antigens such as NspA [47]. Thus a method may use an OMVextraction buffer with about 0.5% deoxycholate or lower e.g. about 0.2%,about 0.1%, <0.05% or zero.

A useful process for OMV preparation is described in reference 51 andinvolves ultrafiltration on crude OMVs, rather than instead of highspeed centrifugation. The process may involve a step ofultracentrifugation after the ultrafiltration takes place.

The invention provides an OMV obtained or obtainable by the methodsdescribed above.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising (a) anOMV of the invention and (b) a pharmaceutically acceptable carrier. Theinvention also provides a process for preparing such a composition,comprising the step of admixing OMVs of the invention with apharmaceutically acceptable carrier.

The invention also provides a container (e.g. vial) or delivery device(e.g. syringe) pre-filled with a pharmaceutical composition of theinvention. The invention also provides a process for providing such acontainer or device, comprising introducing into the container or devicea vesicle-containing composition of the invention.

The immunogenic composition may include a pharmaceutically acceptablecarrier, which can be any substance that does not itself induce theproduction of antibodies harmful to the patient receiving thecomposition, and which can be administered without undue toxicity.Pharmaceutically acceptable carriers can include liquids such as water,saline, glycerol and ethanol. Auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, and the like, can also bepresent in such vehicles. A thorough discussion of suitable carriers isavailable in ref 52.

Bacteria can affect various areas of the body and so the compositions ofthe invention may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The compositionmay be prepared for topical administration e.g. as an ointment, cream orpowder. The composition be prepared for oral administration e.g. as atablet or capsule, or as a syrup (optionally flavoured). The compositionmay be prepared for pulmonary administration e.g. as an inhaler, using afine powder or a spray. The composition may be prepared as a suppositoryor pessary. The composition may be prepared for nasal, aural or ocularadministration e.g. as drops.

A pharmaceutical carrier may include a temperature protective agent, andthis component may be particularly useful in adjuvanted compositions(particularly those containing a mineral adjuvant, such as an aluminiumsalt). As described in reference 53, a liquid temperature protectiveagent may be added to an aqueous vaccine composition to lower itsfreezing point e.g. to reduce the freezing point to below 0° C. Thus thecomposition can be stored below 0° C., but above its freezing point, toinhibit thermal breakdown. The temperature protective agent also permitsfreezing of the composition while protecting mineral salt adjuvantsagainst agglomeration or sedimentation after freezing and thawing, andmay also protect the composition at elevated temperatures e.g. above 40°C. A starting aqueous vaccine and the liquid temperature protectiveagent may be mixed such that the liquid temperature protective agentforms from 1-80% by volume of the final mixture. Suitable temperatureprotective agents should be safe for human administration, readilymiscible/soluble in water, and should not damage other components (e.g.antigen and adjuvant) in the composition. Examples include glycerin,propylene glycol, and/or polyethylene glycol (PEG). Suitable PEGs mayhave an average molecular weight ranging from 200-20,000 Da. In apreferred embodiment, the polyethylene glycol can have an averagemolecular weight of about 300 Da (‘PEG-300’).

The composition is preferably sterile. It is preferably pyrogen-free. Itis preferably buffered e.g. at between pH 6 and pH 8, generally aroundpH 7. Compositions of the invention may be isotonic with respect tohumans.

Immunogenic compositions comprise an immunologically effective amount ofimmunogenic vesicles, as well as any other of other specifiedcomponents, as needed. By ‘immunologically effective amount’, it ismeant that the administration of that amount to an individual, either ina single dose or as part of a series, is effective for treatment orprevention. This amount varies depending upon the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.

Previous work with vesicle vaccines (e.g. for meningococcus) offerspharmaceutical, posological and formulation guidance for compositions ofthe invention. The concentration of vesicles in compositions of theinvention will generally be between 10 and 500 μg/ml, preferably between25 and 200 μg/ml, and more preferably about 50 μg/ml or about 100 μg/ml(expressed in terms of total protein in the vesicles). A dosage volumeof 0.5 ml is typical for injection.

The composition may be administered in conjunction with otherimmunoregulatory agents. Adjuvants which may be used in compositions ofthe invention include, but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref 57], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt.

The adjuvants known as “aluminium hydroxide” are typically aluminiumoxyhydroxide salts, which are usually at least partially crystalline.Aluminium oxyhydroxide, which can be represented by the formula AlO(OH),can be distinguished from other aluminium compounds, such as aluminiumhydroxide Al(OH)₃, by infrared (IR) spectroscopy, in particular by thepresence of an adsorption band at 1070 cm⁻¹ and a strong shoulder at3090-3100 cm⁻¹ [chapter 9 of ref 57]. The degree of crystallinity of analuminium hydroxide adjuvant is reflected by the width of thediffraction band at half height (WHH), with poorly-crystalline particlesshowing greater line broadening due to smaller crystallite sizes. Thesurface area increases as WHH increases, and adjuvants with higher WHHvalues have been seen to have greater capacity for antigen adsorption. Afibrous morphology (e.g. as seen in transmission electron micrographs)is typical for aluminium hydroxide adjuvants. The pI of aluminiumhydroxide adjuvants is typically about 11 i.e. the adjuvant itself has apositive surface charge at physiological pH. Adsorptive capacities ofbetween 1.8-2.6 mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported foraluminium hydroxide adjuvants.

The adjuvants known as “aluminium phosphate” are typically aluminiumhydroxyphosphates, often also containing a small amount of sulfate (i.e.aluminium hydroxyphosphate sulfate). They may be obtained byprecipitation, and the reaction conditions and concentrations duringprecipitation influence the degree of substitution of phosphate forhydroxyl in the salt. Hydroxyphosphates generally have a PO₄/Al molarratio between 0.3 and 1.2. Hydroxyphosphates can be distinguished fromstrict AlPO₄ by the presence of hydroxyl groups. For example, an IRspectrum band at 3164 cm⁻¹ (e.g. at 200° C.) indicates the presence ofstructural hydroxyls [ch. 9 of ref 57].

The PO₄/Al³⁺ molar ratio of an aluminium phosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, andmore preferably 0.95±0.1. The aluminium phosphate will generally beamorphous, particularly for hydroxyphosphate salts. A typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at 0.6 mg Al³⁺/ml. The aluminium phosphate willgenerally be particulate (e.g. plate-like morphology as seen intransmission electron micrographs). Typical diameters of the particlesare in the range 0.5-20 μm (e.g. about 5-10 μm) after any antigenadsorption. Adsorptive capacities of between 0.7-1.5 mg protein per mgAl⁺⁺⁺ at pH 7.4 have been reported for aluminium phosphate adjuvants.

The point of zero charge (PZC) of aluminium phosphate is inverselyrelated to the degree of substitution of phosphate for hydroxyl, andthis degree of substitution can vary depending on reaction conditionsand concentration of reactants used for preparing the salt byprecipitation. PZC is also altered by changing the concentration of freephosphate ions in solution (more phosphate=more acidic PZC) or by addinga buffer such as a histidine buffer (makes PZC more basic). Aluminiumphosphates used according to the invention will generally have a PZC ofbetween 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.

Suspensions of aluminium salts used to prepare compositions of theinvention may contain a buffer (e.g.

a phosphate or a histidine or a Tris buffer), but this is not alwaysnecessary. The suspensions are preferably sterile and pyrogen-free. Asuspension may include free aqueous phosphate ions e.g. present at aconcentration between 1.0 and 20 mM, preferably between 5 and 15 mM, andmore preferably about 10 mM. The suspensions may also comprise sodiumchloride.

In one embodiment, an adjuvant component includes a mixture of both analuminium hydroxide and an aluminium phosphate. In this case there maybe more aluminium phosphate than hydroxide e.g. a weight ratio of atleast 2:1 e.g. ≧5:1, ≧6:1, ≧7:1, ≧8:1, ≧9:1, etc.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 10 mg/ml e.g. ≦5 mg/ml, ≦4 mg/ml, ≦3mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc. A preferred range is between 0.3 and 1mg/ml. A maximum of <0.85 mg/dose is preferred.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 [Chapter 10 of ref 57;see also ref 54] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

Various suitable oil-in-water emulsions are known, and they typicallyinclude at least one oil and at least one surfactant, with the oil(s)and surfactant(s) being biodegradable (metabolisable) and biocompatible.The oil droplets in the emulsion are generally less than 5 μm indiameter, and advantageously the emulsion comprises oil droplets with asub-micron diameter, with these small sizes being achieved with amicrofluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm are preferred as they can be subjected to filtersterilization.

The invention can be used with oils such as those from an animal (suchas fish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Otherpreferred oils are the tocopherols (see below). Oil in water emulsionscomprising squalene are particularly preferred. Mixtures of oils can beused.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brijsurfactants), such as triethyleneglycol monolauryl ether (Brij 30); andsorbitan esters (commonly known as the SPANs), such as sorbitantrioleate (Span 85) and sorbitan monolaurate. Preferred surfactants forincluding in the emulsion are Tween 80 (polyoxyethylene sorbitanmonooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. Asmentioned above, detergents such as Tween 80 may contribute to thethermal stability seen in the examples below.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

A submicron emulsion of squalene, Tween 80, and Span 85. The compositionof the emulsion by volume can be about 5% squalene, about 0.5%polysorbate 80 and about 0.5% Span 85. In weight terms, these ratiosbecome 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. Thisadjuvant is known as ‘MF59’ [54-56], as described in more detail inChapter 10 of ref. 57 and chapter 12 of ref. 58. The MF59 emulsionadvantageously includes citrate ions e.g. 10 mM sodium citrate buffer.

An emulsion comprising squalene, an α-tocopherol, and polysorbate 80.These emulsions may have from 2 to 10% squalene, from 2 to 10%tocopherol and from 0.3 to 3% Tween 80, and the weight ratio ofsqualene:tocopherol is preferably ≦1 (e.g. 0.90) as this provides a morestable emulsion. Squalene and Tween 80 may be present volume ratio ofabout 5:2, or at a weight ratio of about 11:5. One such emulsion can bemade by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 mlsqualene), then microfluidising the mixture. The resulting emulsion mayhave submicron oil droplets e.g. with an average diameter of between 100and 250 nm, preferably about 180 nm.

An emulsion of squalene, a tocopherol, and a Triton detergent (e.g.Triton X-100). The emulsion may also include a 3d-MPL (see below). Theemulsion may contain a phosphate buffer.

An emulsion comprising a polysorbate (e.g. polysorbate 80), a Tritondetergent (e.g. Triton X-100) and a tocopherol (e.g. an α-tocopherolsuccinate). The emulsion may include these three components at a massratio of about 75:11:10 (e.g. 750 μg/ml polysorbate 80, 110 μg/ml TritonX-100 and 100 μg/ml α-tocopherol succinate), and these concentrationsshould include any contribution of these components from antigens. Theemulsion may also include squalene. The emulsion may also include a3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

An emulsion of squalane, polysorbate 80 and poloxamer 401 (“Pluronic™L121”). The emulsion can be formulated in phosphate buffered saline, pH7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides,and has been used with threonyl-MDP in the “SAF-1” adjuvant [59](0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate80). It can also be used without the Thr-MDP, as in the “AF” adjuvant[60] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).Microfluidisation is preferred.

An emulsion comprising squalene, an aqueous solvent, a polyoxyethylenealkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12)cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. asorbitan ester or mannide ester, such as sorbitan monoleate or ‘Span80’). The emulsion is preferably thermoreversible and/or has at least90% of the oil droplets (by volume) with a size less than 200 nm [61].The emulsion may also include one or more of: alditol; a cryoprotectiveagent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or analkylpolyglycoside. Such emulsions may be lyophilized.

An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid,and 0.05-5% of a non-ionic surfactant. As described in reference 62,preferred phospholipid components are phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin.Submicron droplet sizes are advantageous.

A submicron oil-in-water emulsion of a non-metabolisable oil (such aslight mineral oil) and at least one surfactant (such as lecithin, Tween80 or Span 80). Additives may be included, such as QuilA saponin,cholesterol, a saponin-lipophile conjugate (such as GPI-0100, describedin reference 63, produced by addition of aliphatic amine todesacylsaponin via the carboxyl group of glucuronic acid),dimethyidioctadecylammonium bromide and/orN,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine.

An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylatedfatty alcohol, and a non-ionic hydrophilic surfactant (e.g. anethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene blockcopolymer) [64].

An emulsion comprising a mineral oil, a non-ionic hydrophilicethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g.an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropyleneblock copolymer) [64].

An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. acholesterol) are associated as helical micelles [65].

Antigens and adjuvants in a composition will typically be in admixtureat the time of delivery to a patient. The emulsions may be mixed withantigen during manufacture, or extemporaneously, at the time ofdelivery. Thus the adjuvant and antigen may be kept separately in apackaged or distributed vaccine, ready for final formulation at the timeof use. The antigen will generally be in an aqueous form, such that thevaccine is finally prepared by mixing two liquids. The volume ratio ofthe two liquids for mixing can vary (e.g. between 5:1 and 1:5) but isgenerally about 1:1.

C. Saponin Formulations [Chapter 22 of Ref 57]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterogeneous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree has been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref 66.Saponin formulations may also comprise a sterol, such as cholesterol[67].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexs (ISCOMs; see chapter 23 ofref 57; also refs 68 & 69). ISCOMs typically also include a phospholipidsuch as phosphatidylethanolamine or phosphatidylcholine. Any knownsaponin can be used in ISCOMs. Preferably, the ISCOM includes one ormore of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid ofadditional detergent [70].

A review of the development of saponin based adjuvants can be found inrefs. 71 & 72.

D. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref 73. Such “small particles” of 3dMPL are small enough tobe sterile filtered through a 0.22 μm membrane [73]. Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC-529 [74,75].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 76 & 77.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 78, 79 and 80 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 81-86.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [87]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 88-90. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 91-93.

A particularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC-31™ [94-96]. Thus an adjuvant used withthe invention may comprise a mixture of (i) an oligonucleotide (e.g.between 15-40 nucleotides) including at least one (and preferablymultiple) CpI motifs (i.e. a cytosine linked to an inosine to form adinucleotide), and (ii) a polycationic polymer, such as an oligopeptide(e.g. between 5-20 amino acids) including at least one (and preferablymultiple) Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may bea deoxynucleotide comprising 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID NO:2). The polycationic polymer may be a peptide comprising 11-mer aminoacid sequence KLKLLLLLKLK (SEQ ID NO: 3). This combination of SEQ IDNOs: 6 and 7 provides the IC-31™ adjuvant.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref 97 and as parenteraladjuvants in ref 98. The toxin or toxoid is preferably in the form of aholotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 99-106. A useful CT mutant is or CT-E29H[107]. Numerical reference for amino acid substitutions is preferablybased on the alignments of the A and B subunits of ADP-ribosylatingtoxins set forth in ref 108, specifically incorporated herein byreference in its entirety.

E. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [109], etc.) [110], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor. Apreferred immunomodulator is IL-12.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [111] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [112].

G. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes (Chapters 13 & 14 of ref. 57)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 113-115.

I. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 116 and 117.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [118]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [119]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL)+a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [120]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [121]; (6) SAF,containing 10% squalane, 0.4% Tween 80™, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion. (7) Ribi™ adjuvant system(RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); and (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref 57.

An aluminium hydroxide adjuvant is useful, and antigens are generallyadsorbed to this salt. Oil-in-water emulsions comprising squalene, withsubmicron oil droplets, are also preferred, particularly in the elderly.Useful adjuvant combinations include combinations of Th1 and Th2adjuvants such as CpG & an aluminium salt, or resiquimod & an aluminiumsalt. A combination of an aluminium salt and 3dMPL may be used.

Immunisation

In addition to providing immunogenic compositions as described above,the invention also provides a method for raising an immune response in amammal, comprising administering an immunogenic composition of theinvention to the mammal Typically, the immune response is an antibodyresponse. The antibody response is preferably a protective antibodyresponse. The invention also provides compositions of the invention foruse in such methods.

The invention also provides a method for protecting a mammal against abacterial infection and/or disease, comprising administering to themammal an immunogenic composition of the invention.

The invention provides compositions of the invention for use asmedicaments (e.g. as immunogenic compositions or as vaccines). It alsoprovides the use of OMVs of the invention in the manufacture of amedicament for preventing a bacterial infection in a mammal.

The mammal is preferably a human. The human may be an adult or,preferably, a child. Where the vaccine is for prophylactic use, thehuman is preferably a child (e.g. a toddler or infant); where thevaccine is for therapeutic use, the human is preferably an adult. Avaccine intended for children may also be administered to adults e.g. toassess safety, dosage, immunogenicity, etc.

Efficacy of therapeutic treatment can be tested by monitoring bacterialinfection after administration of the composition of the invention.Efficacy of prophylactic treatment can be tested by monitoring immuneresponses against immunogenic proteins in the vesicles or other antigensafter administration of the composition. Immunogenicity of compositionsof the invention can be determined by administering them to testsubjects (e.g. children 12-16 months age) and then determining standardserological parameters. These immune responses will generally bedetermined around 4 weeks after administration of the composition, andcompared to values determined before administration of the composition.Where more than one dose of the composition is administered, more thanone post-administration determination may be made.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is about 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Identity between polypeptide sequences is preferably determined by theSmith-Waterman homology search algorithm as implemented in the MPSRCHprogram (Oxford Molecular), using an affine gap search with parametersgap open penalty=12 and gap extension penalty=1.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a map of pET-OmpA plasmid, which is a pET21b-derivativeplasmid containing the nucleic acid sequence encoding the E. coli OmpAsignal sequence (SS) fused to a gene of interest (GOI) encoding aheterologous protein. The OmpA LS targets the protein encoded by the GOIinto the lumen of OMVs.

FIG. 2 shows the results of SDS-polyacrylamide gel electrophoresis(SDS-PAGE) of total lysates of cultures before and after induction with1 mM IPTG. Bands corresponding to SpyCEP, Slo, Bla, fHbp and Spy0269 arehighlighted by arrows.

FIG. 3 shows an SDS-PAGE of 30 μg OMVs prepared by ultracentrifugationof culture supernatant of the transformed ΔtolR and ΔompA strains. Bandscorresponding to the weight of SpyCEP, Slo, Bla and fHbp are indicatedby arrows.

FIG. 4 shows the results from the Western blots for Slo (A) and SpyCEP(B). Empty OMVs were also loaded as a negative control. Comparing thechemiluminescence signals with those from the known amounts of purifiedproteins, demonstrated that 30 μg of OMVs contain approximately 240 ngSlo-dm, 240 ng SpyCEP.

FIG. 5 shows the results from the Western blots for Bla (A) and fHbp(B). Empty OMVs were also loaded as a negative control. Comparing thechemiluminescence signals with those from the known amounts of purifiedproteins, demonstrated that 30 μg of OMVs contain approximately 240 ngBla.

FIG. 6 shows Western blots showing that the Slo and SpyCEP are expressedas luminal components of OMVs, rather than attached to theirextracellular surface. 100 μg/ml proteinase K was added to 15 μg intactand solubilized (in 1% SDS) vesicles expressing Slo or SpyCEP andincubated at 37° C. 10 minutes. Protein degradation was detected byWestern blot analysis.

FIG. 7 shows a Western blot showing that Bla is expressed as a luminalcomponent of OMVs, rather than attached to their extracellular surface.100 μg/ml proteinase K was added to 15 μg intact and solubilized (in 1%SDS) OMVs expressing Bla and incubated at 37° C. 10 minutes. Proteindegradation was detected by Western blot analysis.

FIG. 8A shows SpyCEP activity of SpyCEP-containing OMVs which have beensolubilized with 0.5% Triton X-100. OMVs expressing the SpyCep proteinwere incubated at different concentration with 50 μg/ml IL-8 at 37° C.for 2 hours. SpyCep wild type protein was used as positive control at 10μg/ml and hydrolysis of IL-8 was analyzed by SDS-PAGE.

FIG. 8B shows SpyCEP activity of SpyCEP-containing OMVs which have beensolubilized with 1% Triton X-100. OMVs expressing the SpyCep proteinwere incubated at different concentration with 50 μg/ml IL-8 at 37° C.for 2 hours. SpyCep wild type protein was used as positive control at 10μg/ml and hydrolysis of IL-8 was analyzed by SDS-PAGE.

FIG. 9A shows the Bla activity of Bla-containing OMVs and empty OMVs.OMV preparations were incubated with nitrocefin (0.5 mg/ml; Oxoid,Thermo Scientific, Cambridge, United Kingdom) for 30 min at 37° C. inthe dark. The chromogen hydrolysis and subsequent color change ofsupernatants were determined immediately with the Tecanspectrophotometer at OD₄₈₅. The enzymatic activity was estimated using astandard curve, where OD₄₈₅ was related to the amount of nitrocefinhydrolyzed. This was quantified using recombinant β-lactamase (VWR).

FIG. 9B shows the hemolytic activity, expressed as is the ratio betweenthe absorbance (OD 540 nm) of blood incubated with OMVs and theabsorbance of blood incubated with water (100% hemolysis), of wild type(wt) Slo-containing OMVs and empty OMVs when incubated with sheep blooderythrocytes.

FIG. 10 shows the geometric mean of the ELISA titers obtained from the 8mice for each group of immunizations after the third immunization (day49).

FIG. 11 shows the percentage amount of uncleaved IL-8 in the presence of100 ng of recombinant SpyCEP and different dilutions of immune seracollected after 3 (post 3) immunization as determined by the ELISAassay.

FIG. 12A shows the percentage amount of uncleaved IL-8 in the presenceof 100 ng of recombinant SpyCEP and different dilutions of immune seracollected after 2 (post 2) immunization as determined by the ELISAassay.

FIG. 12B shows the percentage amount of uncleaved IL-8 in the presenceof recombinant SpyCEP or different dilutions of immune sera collectedafter 2 (post 2) immunization with SpyCep containing OMVs or empty OMVand SpyCep as determined by the ELISA assay. PBS was used as a negativecontrol.

FIG. 13 shows the percentage amount of haemolysis in the presence ofdifferent dilutions of immune sera collected from mice immunized withrecombinant Slo, as a positive control, or with engineered OMVscontaining Slo (OMV-Slo).

FIG. 14 shows a survival plot of samples of 8 mice immunized with OMVscarrying Slo and SpyCEP, with or without sonication before absorption toAlum. The survival plots of mice immunized with recombinant Slo andSpyCEP absorbed to Alum are used as a control. FIG. 15A shows a map ofthe pET-slo+spy0269 plasmid.

FIG. 15B shows a map of the pET-slo+spy0269+spycep plasmid.

FIG. 16A shows Western blots showing that all of the proteins areexpressed from the bi-cistronic and the tri-cistronic constructs afterinduction with IPTG.

FIG. 16B shows Western blots showing that both Slo and Spy0269 proteinsare expressed and present into the OMVs.

MODES FOR CARRYING OUT THE INVENTION Example 1 Expression ofHeterologous Proteins into E. coli OMVs

Generation of E. coli BL21(DE3) ΔtolR and ΔompA ko Mutants

Recombination-prone BL21(DE3) cells were produced by using the highlyproficient homologous recombination system (red operon) [122]. Briefly,electrocompetent bacterial cells were transformed with 5 μg of plasmidpAJD434 by electroporation (5.9 ms at 2.5 kV). Bacteria were then grownfor 1 h at 37° C. in 1 ml of SOC broth and then plated on Luria-Bertani(LB) plates containing trimethoprim (100 μg/ml). Expression of the redgenes carried by pAJD434 was induced by adding 0.2% L-arabinose to themedium.

ΔtolR and ΔompA E. coli BL21 mutant strains, which are known tospontaneously produce a large amount of OMVs, were produced by replacingompA and tolR coding sequences with kanamycin (kmr) and chloramphenicol(cmr) resistance cassettes, respectively. A three-step PCR protocol wasused to fuse the upstream and downstream regions of ompA and tolR to thekmr and cmr genes, respectively. Briefly, the upstream and downstreamregions of the tolR and ompA gene were amplified from BL21(DE3) genomicDNA with the specific primer pairs tolR-1/tolR-2 and tolR-3/tolR-4;ompA-1/ompA-2 and ompA-3/ompA 4, respectively (Table 1). The kmrcassette was amplified from plasmid pUC4K using the primers PUC4K-revand PUC4K-for and cmr was amplified using primers CMR-for/CMR-rev.Finally, 100 ng of each of the three amplified fragments were fusedtogether by mixing in a PCR containing the ¼ primers.

Linear fragments in which the antibiotic resistance gene was flanked bythe tolR/ompA upstream and downstream regions were used to transform therecombination-prone BL21(DE3) E. coli strain, which was madeelectrocompetent by three washing steps in cold water. Transformationwas by an electroporation of 5.9 ms at 2.5 kV. Transformants wereselected by plating the cells on LB plates containing 30 μg/ml ofkanamycin or 20 μg/ml chloramphenicol. The deletion of the tolR and ompAgenes was confirmed by PCR-amplification of genomic DNA using primerspairs tolR-1/PUC4K-rev and PUC4K-for/tolR-4; ompA-1/CMR-rev andCMR-for/ompA-4.

TABLE 1 Oligonucleotide primers: SEQ ID Name Sequence NO GAS25-FACCGTAGCGCAGGCCAACAAACAAAACACTGCTAGTACAG 4 GAS25-RGTGATGGTGATGTTACTACTTATAAGTAATCGAACCATATG 5 SpyCEP-F3ACCGTAGCGCAGGCCGCAGCAGATGAGCTAAGCACAATGAGCGAACC 6 SpyCEP-R3GTGATGGTGATGTTATTAGGCTTTTGCTGTTGCTGAGGTCGTTGAC 7 TTGGTTGG Bla-omp-FACCGTAGCGCAGGCCCGGTAAGATCCTTGAGATTTTTCG 8 Bla-omp-RGTGATGGTGATGTTATTACCAATGCTTAATCAGTGAGGC 9 fHbp-FACCGTAGCGCAGGCCGTCGCCGCCGACATCG 10 fHbp-RGTGATGGTGATGTTATTATTGCTTGGCGGCAAGGC 11 omprev GGCCTGCGCTACGGTAGCGAAA 12nohisflag TAACATCACCATCACCATCACGATTACAAAGA 13 tolR-1TCTGGAATCGAACTCTCTCG 14 tolR-2ATTTTGAGACACAACGTGGCTTTCATGGCTTACCCCTTGTTG 15 tolR-3TTCACGAGGCAGACCTCATAAACATCTGCGTTTCCCTTG 16 tolR-4 TTGCTTCTGCTTTAACTCGG17 ompA-1 GATCGGTTGGTTGGCAGAT 18 ompA-2CACCAGGATTTATTTATTCTGCGTTTTTGCGCCTCGTTATCAT 19 ompA-3TACTGCGATGAGTGGCAGGCGCAGGCTTAAGTTCTCGTC 20 ompA-4 AAAATCTTGAAAGCGGTTGG21 PUC4K-rev AAAGCCACGTTGTGTCTC 22 PUC4K-for TGAGGTCTGCCTCGTGAA 23CMR-for CGCAGAATAAATAAATCCTGGTG 24 CMR-rev CCTGCCACTCATCGCAGTA 25Spy0269-F ACCGTAGCGCAGGCCGATGATAGAGCCTCAGGAGAAACG 26 Spy0269-RGTGATGGTGATGTTATCACTTAGATTCCTTACGGAACC 27 Spy0269-GATTACTTATAAGTAGAGAAGGAGATATACATATGAAAAAGACA 28 fus3 GC Slo-fus-FAACAAACAAAACACTGCTAGTACAG 29 Slo-fus-R3TATACTCCTTCTCTACTTATAAGTAATCGAACCATATG 30 Spy0269-fus-TCACTTAGATTCCTTACGGAACC 31 R Spycep-fus-FAAGGAATCTAAGTGAGAAGGAGATATACATATGAAAAAGACAGC 32 AGTGAGA

Example 2 Plasmid Construction

Five heterologous proteins from different bacterial species, both Grampositive and Gram-negative, and belonging to different cellularcompartments were selected as model proteins to determine whetherheterologous proteins can be incorporated into E. coli OMVs in theirnative conformations. These proteins included: (1) the periplasmic TEM1beta lactamase (Bla) from E. coli, (2) the factor H-binding protein(fHbp) lipoprotein from Neisseria meningitidis, (3) the extracellularcholesterol depending streptolysin O (Slo also called GAS25) fromStreptococcus pyogenes, (4) the cell envelope serine protease SpyCEP(also called GAS57) from Streptococcus pyogenes and (5) the putativesurface exclusion protein Spy0269 (also known GAS40) also fromStreptococcus pyogenes. The nucleic acid coding sequences for each ofthese five proteins were cloned into the pET-OmpA plasmid using thepolymerase incomplete primer extension (PIPE) cloning method [123]. ThepET-OmpA plasmid (as provided in e.g. [124] and [125]) is apET21b-derived plasmid.

Briefly, Slo-dm (slo double mutant, SEQ ID:42) was PCR-amplified fromplasmid pET21-Slo-dm, which contains the slo-dm gene, using theGAS25-F/GAS25-R primers (see Table 1). The SpyCEP gene (a double mutantsequence is also provided as SEQ ID:40) was PCR-amplified from the M1GAS strain ISS3348 using SpyCEP-F3/SpyCEP-R3 primers, which weredesigned to exclude the C-terminal LPXTG motif cell-wall anchor (locatedat aa 1614-1647). The spy0269 (SEQ ID:44) gene was PCR amplified fromthe M1 GAS strain ISS3348 using spy0269-F/spy0269-R primers. Primers forfHbp gene amplification were designed to exclude the lipobox (which islocated at aa 17-25 in fHbp) in order to avoid membrane anchoring. Inorder to target the proteins to the periplasm, the sequences encodingthe signal sequences of each of these proteins was removed and replacedwith the E. coli OmpA signal sequence (SS) (see FIG. 1). Bla and fHbpwere amplified from pET21b and Neisseria meningitidis MC58 genomerespectively using primers Bla-omp-F/Bla-omp-R and fHbp-F/fHbp-R,respectively. pET-OmpA plasmid was amplified by PCR using primersomprev/nohisflag. In this way plasmids pET-21_Bla (SEQ ID:37),pET-21_slo (SEQ ID:33), pET-21_SpyCEP (SEQ ID:34), pET-21_fHbp (SEQID:36) and pET21_spy0269 (SEQ ID:35) were generated.

Example 3 Expression of the Heterologous Proteins into ΔtolR and ΔompAMutants, OMVs and Total Lysates Preparation

In order to investigate whether the Bla, slo, SpyCEP, fHbp and Spy0269proteins are packaged into OMVs, the ΔtolR and ΔompA E. coli BL21strains were transformed with the pET-21_Bla, pET-21_slo, pET-21_SpyCEP,pET-21_fHbp plasmids and pET21_spy0269. As a negative control, the ΔtolRand ΔompA E. coli BL21 strains were transformed with the pET-OmpA emptyvector.

All the strains were grown in liquid cultures until logarithmic phaseand induction of expression of the genes was carried out by adding 1 mMIPTG (isopropyl-beta-D-thiogalactopyranoside). FIG. 2 shows theSDS-polyacrylamide gel electrophoresis (SDS-PAGE) of total lysates ofthese cultures before and after induction with 1 mM IPTG. Bandscorresponding to Bla, slo, SpyCEP, fHbp and Spy0269 proteins are presentin all induced samples and are indicated by arrows. Thus, all five ofthe tested heterologous proteins were successfully induced in E. coli.

Using an overnight culture of each transformant, 200 ml of LB medium wasinoculated at OD₆₀₀=0.05. Cultures were grown until the OD₆₀₀=0.5 andthen expression of the recombinant proteins was induced by addition of 1mM IPTG, followed by a further incubation of 2 hours. For OMVpreparations, cells were harvested by centrifugation at 8,000×g for 20minutes. The resulting supernatant was filtered through a 0.22 μm poresize filter (Millipore). The filtrates were then subjected to high speedcentrifugation (200,000×g for 2 hours) and the pellets containing theOMVs were resuspended in PBS with protease inhibitors (Roche). For miceimmunization, when indicated, OMVs were sonicated in a hypotonic buffer(10 mM Tris pH 7.5, 1.5 mM MgCl₂, 10 mM KCl) by 10 bursts of 30 secondseach, followed by cooling on ice. Total lysates were prepared from 1 mlof culture, which was centrifuged at 13,000×g for 5 minutes. The pelletwas resuspended in SDS-PAGE sample loading buffer, heated at 100° C. for5 minutes and loaded onto a 4-12% polyacrylamide gel (Invitrogen). Gelswere run in MES buffer (Invitrogen) and stained with Comassie Blue. FIG.3 shows the polyacrylamide gels for approximately 30 mg OMVs obtainedfrom ΔtolR and ΔompA strains which contain plasmids to express thedifferent heterologous proteins. Bands corresponding to the weight ofSpyCEP, Slo, Bla and fHbp are indicated by arrows. To confirm proteinidentification, the bands were excised and digested with trypsin and theresulting proteolytic peptides were then analysed by matrix-assistedlaser desorption ionization—time of flight (MALDI-TOF).

Briefly, protein bands were excised from the gels, washed with 50 mMammonium bicarbonate-acetonitrile (50/50, vol/vol), and air dried. Driedspots were digested for 2 h at 37° C. in 12 μl of 0.012-μg/μlsequencing-grade modified trypsin (sequencing grade modified porcinetrypsin; Promega, Madison, Wis.) in 5 mM ammonium bicarbonate. Afterdigestion, 0.6 μl of the digested product was loaded on amatrix-prespotted Anchorchip (PAC 384 HCCA; Bruker-Daltonics, Bremen,Germany) and air-dried. Spots were washed with 0.6 μl of a solutioncontaining 70% ethanol and 0.1% trifluoroacetic acid. Mass spectra wereacquired with an ultraflex MALDI-TOF mass spectrometer(Bruker-Daltonics). Spectra were externally calibrated by using thecombination of standards present on the PAC chip (Bruker-Daltonics).Monoisotopic peptide matching and protein searching were performedautomatically using a licensed version of the MASCOT software (MatrixSciences, London, United Kingdom) run on a local database. The MASCOTsearch parameters used were as follows: (i) allowed number of missedcleavages=1; (ii) variable posttranslational modification=methionineoxidation; and (iii) peptide tolerance=100 ppm. Only significant hits,as defined by MASCOT probability analysis, were considered. The MASCOTsoftware identified the bands as corresponding to Bla, Slo, SpyCEP andfHbp proteins respectively. These results confirm that Bla, Slo, SpyCEPand fHbp can all be expressed and incorporated into OMVs produced bothby ΔtolR and ΔompA E. coli strains.

Example 4 Quantification of Heterologous Proteins into OMVs

In order to quantify the amount of heterologous proteins incorporated inthe E. coli OMVs, Western blot analysis was performed.

30 μg OMVs containing the heterologous proteins were loaded onto 4-12%SDS-polyacrylamide gels along with increasing concentration (20-80 ng)of the corresponding purified protein. Empty OMVs were also loaded as anegative control. The polyacrylamide gels were then transferred ontonitrocellulose filter by standard methods [126]. The filters wereblocked overnight at 4° C. by agitation in blocking solution (10%skimmed milk and 0.05% Tween in PBS), followed by incubation for 90minutes at 37° C. with a 1:1000 dilution of the required antibody serum(anti-Bla (Abeam), anti-slo, anti-SpyCEP and anti-fHbp) in 3% skimmedmilk and 0.05% Tween in PBS. After three washing steps in PBS-Tween, thefilters were incubated in a 1:2000 dilution of peroxidase-conjugatedanti-mouse immunoglobulin (Dako) in 3% skimmed milk and 0.05% Tween inPBS for an hour, and after three washing steps, the resulting signal wasdetected by using the SuperSignal West Pico chemiluminescent substrate(Pierce).

FIG. 4 and FIG. 5 show the results from the four different Western blotsperformed. As expected from the MALDI-TOF analysis, all the OMVpreparations contain the heterologous proteins selected. Comparing thechemiluminescence signals with those from the known amounts of purifiedproteins, demonstrated that 30 mg of OMVs contain approximately 240 ngof Slo, SpyCEP and Bla.

Example 5 Heterologous Proteins are Localized in the OMVs Periplasm

As described above, the signal sequences of all four of the heterologousproteins were replaced by the E. coli OmpA signal sequence in order totarget the proteins into the E. coli periplasm. It was thereforeimportant to confirm that the proteins are expressed as luminalcomponents of OMVs rather than attached to the extracellular surface ofOMVs.

In order to test this, 100 μg/ml proteinase K (Fermentas) was added to15 μg intact and solubilized (in 1% SDS) OMVs expressing Slo-dm orSpyCEP, and the mixture was then incubated at 37° C. for 10 minutes.After proteinase K deactivation with 10 mM phenylmethylsulfonyl fluoride(PMSF; Sigma Aldrich) samples were loaded on a 4-12% polyacrylamide geland Western blot analysis was performed with the required antibody todetect the presence of the heterologous proteins.

FIG. 6 and FIG. 7 show that Slo-dm, SpyCEP and Bla were all protectedfrom proteinase K-mediated degradation in unsolubilised OMVs (but not insolubilized OMVs), demonstrating than they are both expressed asperiplasmic proteins in the lumen of E. coli OMVs.

Example 6 OMVs Containing SpyCEP are Able to Hydrolyse IL-8

SpyCEP has been reported to hydrolyse IL-8, converting it into a 6-kDainactive fragment [127]. To determine whether SpyCEP maintains thishydrolytic activity in the OMV preparation, OMVs expressing the SpyCEPprotein were incubated at different concentrations with human IL-8 (50μg/ml) at 37° C. for 2 hours. The SpyCEP wild type protein (at aconcentration of 10 μg/ml) was used as positive control. IL-8 wasincubated with 10 ng/ml GAS57 purified protein (which is known tohydrolyse IL-8), as a positive control. The hydrolytic products wereanalysed using an 18% SDS-PAGE with silver staining. In order to testwhether an active form of the SpyCEP protein is located inside the OMVs,leakage of SpyCEP from the OMV lumen was induced by permeabilising theOMVs in 0.5% Triton X-100 at room temperature for 20 minutes (see FIG.8A). An additional experiment was conducted in which the OMVs werepermeabilised using 1% Triton X-100 (see FIG. 8B)

As shown in FIGS. 8A and 8B, IL-8 was almost completely cleaved after 2hours incubation with 30 μg OMVs containing SpyCEP. Thus, SpyCEP'sbiological activity is preserved in OMVs. Retention of functionalactivity indicates that the heterologous protein is correctly folded inthe OMV and will therefore display the same or substantially the samestructural epitopes as the wild-type protein in its native environment.The IL-8 hydrolysis activity was increased in permeabilised OMVs,suggesting that an active form of SpyCEP is located inside OMVs.Retention of functional activity indicates that the heterologous proteinis correctly folded in the OMV and will therefore display the same orsubstantially the same antigens as the wild-type protein in its nativeenvironment.

Example 7 OMVs Containing Bla and Slo

OMVs expressing Bla were incubated with the chromogenic substratenitrocefin and the Bla activity was measured as described herein. OMVpreparations were incubated with nitrocefin (0.5 mg/ml; Oxoid, ThermoScientific, Cambridge, United Kingdom) for 30 min at 37° C. in the dark.The chromogen hydrolysis and subsequent color change of supernatantswere determined immediately with the Tecan spectrophotometer at OD485.The enzymatic activity was estimated using a standard curve, where OD485was related to the amount of nitrocefin hydrolyzed. This was quantifiedusing recombinant β-lactamase (VWR).

FIG. 9A shows that empty OMVs showed no Bla activity, whereas Blaactivity was shown in Bla-containing OMVs. Hence, the biologicalactivity of Bla is preserved in OMVs.

In order to test whether an active form of the Bla is located inside theOMVs, leakage of Bla from the OMV lumen was induced by permeabilisingthe OMVs in 1% Triton X-100 at room temperature for 20 minutes. As shownin FIG. 9A, Bla activity was increased in permeabilised OMVs, suggestingthat an active form of Bla is located inside OMVs.

Slo hemolytic activity was tested by incubating OMVs expressing the wildtype (wt) form of the toxin with sheep blood erythrocytes, using thefollowing method. Serial dilutions of the samples were prepared in96-well plates with U-shaped bottoms using PBS+0.5% BSA as dilutionbuffer. 1 ml of sheep blood was washed three times in PBS (withcentrifugation at 3000×g), and blood cells were finally suspended in 5ml of PBS. 50 μl of this suspension was added to 50 μl of each dilutedsamples and incubated at 37° C. for 30 min. Water was used to give 100%haemolysis, and PBS+BSA 0.5% was used as negative control. Plates werethen centrifuged for 5 min at 1,000×g, and the supernatant was carefullytransferred to 96-well flat-bottomed plates to read the absorbance at540 nm [128].

As shown in FIG. 9B, negative control empty OMVs showed no hemolyticactivity, whereas Slo-wt containing OMVs show high levels of hemolyticactivity, which was dependent on the amount of OMVs present. Hence, thebiological activity of Slo is preserved in OMVs. Retention of functionalactivity of Slo and Bla indicates that these heterologous proteins arecorrectly folded in the OMV and will therefore display the same orsubstantially the same antigens as the wild-type protein in its nativeenvironment.

Example 8 Antibody Titers Elicited in Mice Immunized with Slo and SpyCEPin Engineered OMVs

To examine the immunogenicity of Slo and SpyCEP in E. coli OMVs, groupsof 8 CD1 5-week old female mice were immunized intraperitoneally on days0, 21 and 35 with 25 μg of sonicated or unsonicated OMVs over-expressingthe Slo or SpyCEP proteins. All samples were formulated with 2 mg/mlalum hydroxide as adjuvant. Control mice were immunized with PBS andadjuvant. Positive control groups consisted of mice immunized with 20 μgof recombinant purified Slo or SpyCEP proteins. To test the OMVsadjuvanticity, mice were also immunized with 25 μg of empty OMVs or with25 μg of empty OMVs plus 20 μg of Slo or SpyCEP antigen.

Sera were collected before the first immunization (pre-immune sera) andafter each of the 3 immunizations (post1, post2 and post3 sera), andELISA titers were analysed. ELISAs were performed using 96-well Maxisorpplates (Nunc, Thermo Fisher Scientific) coated with 3 μg/ml or 2 μg/mlof Slo or SpyCEP protein, respectively, in PBS. Plates were incubatedfor 2 h at room temperature, then washed three times with TPBS (0.05%Tween 20 in PBS, pH 7.4) and blocked with 250 μl/well of 2% BSA(Sigma-Aldrich) for 1 h at room temperature. Each incubation step wasfollowed by triple TPBS wash. Serum samples were initially diluted1:500-1:1000 in 2% BSA in TPBS, transferred onto coated-blocked plates(200 μL) and serially diluted (by two-fold) followed by 2 h incubationat 37° C. Then 100 μl/well of 1:2000 diluted alkalinephosphatase-conjugated goat anti-mouse IgG were added and left for 2 hat 30° C. Bound alkaline phosphatase was visualized by adding 100μL/well of 3 mg/mL para-nitrophenyl-phosphate disodium hexahydrate(Sigma-Aldrich) in 1 M diethanolamine buffer (pH 9.8). After 10 min ofdevelopment at room temperature, plates were analysed at 405 nm in amicroplate spectrophotometer. Antibody titres were calculated byinterpolating ODs onto a reference calibration curve, and expressed inELISA units (EU) per mL.

FIG. 10 shows the geometric mean of the ELISA titers obtained from the 8mice for each group of immunizations after the third immunization (day49). Sera from mice immunized with PBS and adjuvant alone or with emptyOMVs gave negative results. As shown in FIG. 10, the antibody responseto all five preparations was statistically equivalent, suggesting thatengineered OMV preparations are able to induce antibody against Slo andSpyCEP antigens.

Example 9 OMV-SpyCEP Immune Serum Inhibits SpyCEP Mediated Processing ofIL-8

Sera obtained from mice immunized with PBS alone, empty OMVs, OMV-SpyCEPand OMV+SpyCEP were tested for their capacity to neutralize IL-8proteolytic activity of SpyCEP. To perform this IL-8 inhibition assay,SpyCEP (0.1 μg/ml) and IL-8 (1 μg/ml) were incubated with pools of micepolyclonal serum from the 8 immunized mice for each group at fivedifferent dilutions (1:2.5, 1:5, 1:10, 1:20, and 1:40) at 37° C. for 2hours in PBS, 0.5 mg/ml BSA. As controls, SpyCEP was incubated withbuffer only, and sera without SpyCEP were used. The amount of uncleavedIL-8 was quantified after 2 h by ELISA (human IL-8 Immunoassay kit,Invitrogen) and expressed as a percentage of uncleaved IL-8 in thereaction (incubation with SpyCEP) compared with IL-8 in the controlreaction (incubation with buffer only).

FIGS. 11, 12A and 12B show the percent of uncleaved IL-8 in the presenceof 100 ng of recombinant SpyCEP and different dilutions of immune seracollected after 3 (post 3) and 2 (post 2) immunizations, respectively,as determined by the ELISA assay.

Sera from mice immunized with recombinant SpyCEP, as positive control,and with engineered OMVs containing SpyCEP (OMV-SpyCEP) were able toneutralize SpyCEP proteolytic activity in a dose-dependent manner andgave statistically equivalent results.

Example 10 OMV-Slo Immune Serum Inhibits Slo Mediated Haemolysis

Sera obtained from mice immunized with PBS alone, empty OMVs, OMV-Slo,20 μg Slo and 0.5 μg Slo (corresponding approximately to the amount ofSlo contained in 20 μg OMVs) were tested for their capacity toneutralize the haemolysis activity of Slo. To perform this assay, Slo(60 ng) was incubated with pooled polyclonal serum from the 8 immunizedmice for each group at six different dilutions (1:31.25; 1:63; 1:125;1:250; 1:500 and 1:1000) for 20 minutes at room temperature in PBS, 0.5mg/ml BSA. 1 ml of sheep blood was washed three times in PBS (withcentrifugation at 3000×g), and the blood cells were finally suspended in5 ml of PBS. 50 μl of this suspension was added to each of the samplesand the samples were incubated at 37° C. for 30 min 60 ng Slo was usedas a positive control to give 100% haemolysis, and PBS+BSA 0.5% was usedas a negative control. Plates were then centrifuged for 5 min at1,000×g, and the supernatant was transferred to 96-well flat-bottomedplates and the absorbance measured at 540 nm [128].

FIG. 13 shows the percent of haemolysis provided by each sample atdifferent sera dilutions. Sera from mice immunized with recombinant Slo,as positive control, and with engineered OMVs containing Slo (OMV-Slo)were able to neutralize the hemolytic activity of Slo in adose-dependent manner and gave statistically equivalent results.

Example 11 OMVs Carrying Foreign Antigens in their Lumen Elicit StrongProtective Responses

Having demonstrated that even if recombinant antigens are present in thelumen of OMVs immunization with such OMVs induce antigen-specificfunctional antibodies, we lastly asked the question whether immunizationcan also induce antigen-specific protective immunity. To test this,female CD1 5-week old mice were immunized intraperitoneally on days 0,21 and 35 with a vaccine formulation including 25 μg of OMV carryingeither Slo or SpyCEP formulated in alum hydroxide. As positive controlsmice were also immunized with 25 μg OMVs carrying Slo and SpyCEPsonicated just before absorption to Alum Hydroxide, and with recombinantSlo, recombinant SpyCEP and recombinant M protein from M1 strain (20 μgeach), all formulated in Alum Hydroxide. Three weeks after the thirdimmunization, mice were infected intraperitoneally with 200 μl of abacterial suspension containing about 2.5E+06 CFU of M1 3348 strain.Mice were monitored on a daily basis for 6 days after treatment andeuthanized when they exhibited defined humane endpoints that had beenpre-established for the study in agreement with Novartis Animal WelfarePolicies.

As shown in the FIG. 14, which reports data from experiments in which 8mice per group were used, OMVs carrying Slo and SpyCEP gave a protectionof 87.5% and 75%, respectively, if not sonicated, very comparable withthe 87.5% and 100% protection values obtained if OMVs were sonicatedbefore absorption to Alum and immunization. This protection also similarto what obtained with recombinant Slo and SpyCEP (81.3% and 87.5%respectively) absorbed to Alum, a remarkable result considering that therecombinant OMVs carry approximately 0.2-0.4 μg of Slo and SpyCEPapproximately 100-fold less than what has been used for the immunizationwith recombinant Slo and SpyCEP.

Example 12 Generation of a Bi-Cistronic Construct for Expression ofSpy0269 and Slo-Dm, and a Tri-Cistronic Construct for Expression ofSpy0269, Slo and SpyCEP, in the OMV Lumen

To generate the bi-cistronic construct, slo-dm and spy0269 genes wereamplified from pET-21_slo and pET-21_spy0269 using slo-fus-F/slo-fus-R3and Spy0269-fus3/Spy0269-R primers respectively. Generated fragmentswere phopshorylated, ligated and cloned into a pET-OmpA plasmid usingthe PIPE cloning method [123] generating a pET-slo+spy0269 plasmid (SEQID:38) (FIG. 15A).

The Slo-dm and Spy0269 proteins were cloned into the pET-OmpA plasmidunder the same T7 promoter, to generate a bi-cistronic construct. AnOmpA leader sequence was cloned upstream of each gene, so that itsexpression was directed to the lumen of the OMVs that were subsequentlyproduced (see below).

To generate the tri-cistronic construct, the pET-spy0269+slo plasmid wasamplified with nohisflag/Spy0269-fus-R primers and spycep gene wasamplified from pET-21 spycep plasmid using spycep-fus-F/spycep-R3primers and cloned into the plasmid using the PIPE cloning method.Generated fragments were phopshorylated, ligated and cloned intopET-OmpA plasmid using the PIPE cloning method [123] generatingpET-slo+spy0269+spycep plasmid (FIG. 15B).

The resulting plasmids were transformed into ΔompA and wild type E. coliBL21 mutant strains for protein induction and OMV preparation.Expression of the cloned genes was induced by adding 1 mM IPTG to thecultures. Western blots were performed on total lysates to verifyprotein expression. FIG. 16A panel I and panel II shows that all of thecloned proteins from the bi-cistronic and the tri-cistronic constructare expressed after induction with IPTG.

OMVs were prepared, as described above, from the ΔompA and wild typestrains containing the bi-cistronic construct. The Western blot resultsof FIG. 16B show that both Slo and Spy0269 proteins are expressed andpresent into the OMVs.

Variant sequences of spy0269 (GAS40) are provided in SEQ IDs:43 to 69.Variant sequences of spyCEP (GAS57) are provided in SEQ IDs:71 to 75 anda detoxified or enzymatically inactive mutant (GAS57 D151A-S617A) isprovided in SEQ IDs:39 and 40. The slo (GAS25) W535F-P427L double mutantis provide in SEQ IDs:41 and 42.

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1. A composition comprising an outer membrane vesicle (OMV) from aGram-negative bacterium and a heterologous protein in the lumen thereof,wherein the heterolobous protein is not integrally associated with themembrane of the OMV; and wherein the composition is capable of elicitingan immune response to the heterologous protein when administered to amammal.
 2. The OMV according to claim 1, wherein the heterologousprotein is a soluble protein.
 3. The composition of claim 1, wherein theheterologous protein is functionally active in the lumen of the OMV. 4.The composition of claim 1, wherein the immune response is an antibodyresponse.
 5. A method for preparing the composition of claim 1, themethod comprising the step of expressing the heterologous protein in theperiplasm of the Gram-negative bacterium.
 6. The method according toclaim 5, wherein the step of expressing comprises an expression vectorcomprising a nucleic acid sequence encoding the heterologous proteinoperatively linked to a nucleic acid encoding a signal sequence of aperiplasmic protein.
 7. The method according to claim 6, wherein the theheterologous protein does not include a native signal sequence.
 8. Themethod of claim 5, further comprising the step of isolating the OMV. 9.An OMV obtained by the method of claim
 5. 10. The composition of claim1, wherein the Gram-negative bacterium is selected from the groupconsisting of E. coli, N. menigitides, Salmonella sp., and Shigella sp.11. The composition of claim 1, wherein the Gram-negative bacterium is ahyperblebbing strain of the Gram-negative bacterium.
 12. The compositionof claim 11, wherein the Gram-negative bacterium is a ΔtolR E. colistrain or a ΔompA E. coli strain.
 13. The composition of claim 1,wherein the heterologous protein is an antigen.
 14. The composition ofclaim 1, wherein the heterologous protein is a cytoplasmic protein or aperiplasmic protein in the heterologous organism.
 15. The composition ofclaim 1, wherein the heterologous protein is a membrane-associatedprotein comprising a membrane anchor in the heterologous organismwherein the membrane anchor is deleted.
 16. The composition of claim 1,wherein the heterologous protein is β lactamase (TEM1), fHbp fromNeisseria meningitides, the double mutant of extracellular cholesteroldepending streptolysin O (Slo-dm) from Streptococcus pyogenes, the cellenvelope serine protease SpyCEP from Streptococcus pyogenes, or theputative surface exclusion protein Spy0269 from Streptococcus pyogenes.17. A pharmaceutical composition comprising (a) the composition of claim1, and (b) a pharmaceutically acceptable carrier.
 18. The pharmaceuticalcomposition according to claim 17, wherein the pharmaceuticalcomposition is a vaccine.
 19. A method of generating an immune responseto a heterologous protein in a mammal, the method comprising a step of:administering the vaccine of claim 18, to the mammal in an amounteffective to elicit an immune response to the heterologous protein. 20.(canceled)